The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.
Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. Affinity chromatography, which exploits a specific interaction between the protein to be purified and an immobilized capture agent, is commonly used for some proteins (e.g., proteins for use as a human therapeutic). Protein A is a useful adsorbent for affinity chromatography of proteins, such as antibodies, which contain an Fc region. Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity (about 10−8 M to human IgG) to the Fc region of antibodies. However, since proteins tend to aggregate or become misfolded, the desired protein (i.e., monomer) is often co-purified with other impurities from these affinity columns, such as protein aggregates, by-products of the cells themselves (i.e., host cell impurities), or virus filter foulant.
Other techniques have been developed to further separate these impurities and mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size, such as ion exchange chromatography, hydrophobic interaction chromatography, or size exclusion chromatography. Several different chromatography resins or sorbents are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long solid phase (e.g., column), achieving a physical separation that increases as they pass further down the solid phase, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. However, each of these methods requires additional buffers, resins or sorbents, and other resources for further purification, and this in turn results in longer processing time and higher cost. Thus, more efficient and economical methods for purifying protein monomers are needed.
Methods of purifying polypeptides from aggregates, multimers, and modified proteins using a protein A column and eluting with a pH gradient elution system was described in U.S. patent application Ser. No. 12/008,160.
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